Two stress proteins of the endoplasmic reticulum bind denatured collagen DEVKINANDAN, ERICH. BALL, AND BISHNUD. SANWAL
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Department of Biochemistry, University of Western Ontario, London, Ont., Canada N6A 5CI Received December 15, 1989 NANDAN,D., BALL,E. H., and SANWAL, B. D. 1990. Two stress proteins of the endoplasmic reticulum bind denatured collagen. Biochem. Cell Biol. 68: 1057-1061. A differentiation-related gelatin-binding 46 kilodalton (kDa) glycoprotein in myoblasts (GP46, colligin) shares several properties with the 78-kDa glucose-regulated protein (GRP78), including location in the endoplasmic reticulum and related C-terminal sequences. These similarities extend to stress inducibility, since we find that GP46 is a heat-shock protein; its synthesis is elevated at 42OC, resulting in a two- to three-fold increase in protein level. Further, GRP78 is a gelatin-binding protein; together with GP46 it is retained on gelatin-Sepharose beads. GRP78 and GP46 do not interact; each protein can be individually eluted, GP46 at low pH and GRP78 by ATP. These results suggest that the proteins have distinct roles in the synthesis of collagen and point to a simple method for purification. Key words: stress proteins, collagen-binding proteins, endoplasmic reticulum, 78-kilodalton glucose-regulated protein, 46-kilodalton glycoprotein.
NANDAN. D., BALL,E. H., et SANWAL,B. D. 1990. Two stress proteins of the endoplasmic reticulum bind denatured collagen. Biochem. Cell Biol. 68 : 1057-1061. Dans les myoblastes, une glycoproteine (GP46, colligine) de 46 kilodaltons (kDa), liant la gklatine et relike a la differenciation, partage plusieurs proprihtks avec la protkine de 78 kDa contr81Ce par le glucose (GRP78), dont la localisation dans le reticulum endoplasmique et les sequences C-terminales apparentees. Ces similitudes comprennent Cgalement l'inductibilite au stress, car nous trouvons que la GP46 est une proteine de choc thermique; a 42°C. sa synthtse elevte augmente le taux protkique de deux a trois fois. De plus, la GRP78 est une protkine liant la gelatine; les deux prottines, GRP78 et GP46, sont retenues sur les billes de gelatine-Stpharose. La GRP78 ne rCagit pas avec la GP46; chacune peut Etre Clute individuellement, la GP46 a faible pH et la GRP78 par I'ATP. Ces rksultats sugghent que ces proteines ont des r6les distincts dans la synthhse du collagtne et laissent prksager une methode simple de purification. Mots cl&s : pprteines de stress, protkines laissent le collagtne, reticulum endoplasmique, proteine de 78 kilodaltons contr81Ce par le glucose, glycoproteine de 46 kilodaltons. [Traduit par la revue]
Eucaryotic cells respond to stress conditions, such as heat shock, glucose deprivation, and certain chemical treatments by overproducing a set of stress proteins (for reviews, see Deshaies et al. 1988; Lee 1987; Pelham 1986). Two major classes of these proteins have been identified: the GRPs are localized to the ER (Pelham 1986; Munro and Pelham 1987) and the HSPs are mostly cytoplasmic (Pelham 1986). This division is not absolute; some GRPs are slightly heat inducible (Lee et al. 1984). Also, an apparent exception with regard to the localization of a HSP is a 47-kDa protein from chicken embryo fibroblasts, which despite its location in the ER has been found to be inducible by heat shock (Nagata et al. 1986). The role of stress proteins is not known for certain, but the consensus is emerging (Pelham 1986) that they participate in the refolding of denatured proteins or the initial folding of newly synthesized proteins. Best understood are GRP78 (also known as BiP) and the related HSP70. In lymphoid cells GRP78 associates with newly synthesized immunoglobulin heavy chains until their assembly with light chains and probably thus prevents the transport of unassembled heavy chains from the ER. In nonlymphoid cells, which do not produce immunoglobulins, GRP78, an inducible protein, associates with abnormal proteins (Lee 1987; Bole et al. 1986; Kozutsumi et al. 1988; Kassenbrock et al. 1988) and probABBREVIATIONS: GP46, 46-kilodalton glycoprotein (colligin); GRP78, 78-kDa glucose-regulated protein; ER, endoplasmic reticulum; HSPs, heat-shock-inducible proteins; BiP, binding protein; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; kDa, kilodalton(s); IgG, immunoglobulin G. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprim6 au Canada
ably helps in their renaturation. Both classes of stress protein probably function to limit damage owing to protein denaturation. While most attention has been devoted to GRP78, next to nothing is known regarding the action of other stress proteins that are present in the lumen of the ER, viz., GRP94 (Lee et al. 1984; Koch 1987) and GP47 (Nagata et al. 1986). During a study of the role of a GP46 in the differentiation of rat skeletal myoblasts (Cates et al. 1984), we discovered that it was localized in the lumen of the ER (Nandan et al. 1988a) and had a RDEL sequence at the C-terminus, as determined by sequencing a partial cDNA clone (Nandan et al. 1988b). A homologous sequence, KDEL, is shared by GRP78 and GRP94 (Munro and Pelham 1987) and may be responsible for retention in the ER. These similarities in structure and location suggested related functions and prompted us to compare them in more detail. In the following report, we demonstrate that in skeletal myoblasts GP46 is a stress protein and GRP78 shares with it the property of binding to gelatin. The functions of the two stress proteins, however, seem to be quite different. Materials and methods Materials A highly myogenic subclone of the rat myoblast cell line, L6, first isolated by Yaffe (1968) was used. A monoclonal antibody against GP46 was obtained as described earlier (Cates et al. 1987). A polyclonal antibody against GRP78 was kindly provided by Dr. H. Pelham (U.K.). Cell culture and metabolic labelling Cells were routinely cultured in minimum essential medium supplemented with 50 pg gentamycin/mL, 16 mM glucose, and 10% horse serum. For tunicamycin treatment and metabolic labelling,
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FIG. 2. Induction of GP46 at 42OC. After 48 h of growth, cultures were transferred to 42OC for various lengths of time. Cells were then lysed in SDS sample buffer, run on SDS gels, and immunoblotted using monoclonal anti-GP46.Odd-numbered lanes are from controls at 37OC; even numbered lanes are from those at 42°C. Lanes 1 and 2, after 6 h of heat treatment; lanes 3 and 4, after 8 h; lanes 5 and 6, after 13 h; lanes 7 and 8, after 24 h. Note that the level of GP46 in control cells increases as the cells approach fusion. (Laemmli 1970). Collagen-Sepharose was made by coupling rat type I collagen to cyanogen bromide activated Sepharose (Pharmacia) according to the manufacturer's instructions.
FIG. 1. Protein synthesis in stressed L6 myoblasts. Cells were treated for 4 h under various conditions: at 37 (lane l), 42 (lane 2), or 44°C (lane 3), or with 0.1 mM sodium arsenate (lane 4), sodium arsenite (lane 5), or 5 p tunicamycin/mL (lane 6). After a further 1 h of labelling with [ 3$ Slmethionine, cultures were lysed in SDS sample buffer and equal amounts of TCA-precipitable radioactivity were loaded on an 8-13% gradient gel. An autoradiograph of the gel is shown. cells were plated at an initial density of 1.5 x 10' cells/60-mm plate and grown for 72 h. After this period, growth medium was replaced with fresh medium containing 5 pg tunicarnycin/mL. After 5 h of preincubation with tunicamycin, cells were labelled for 2 h with 0.1 mCi [35~]methionine/m~ (1 Ci = 36 GBq) in methioninefree medium containing the desired amount of tunicamycin.
Isolation of gelatin- or collagen-binding proteins Cells were rinsed three times with cold PBS and extracted with 2 mL lysis buffer (10 mM Tris-HC1 (pH 7 . 9 , 0.15 M NaCl, 1% Triton X-100, 2 pg leupeptin/mL, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM EGTA). After 5 min on ice, the cells were scraped, passed three times through a 0.5-in 27-gauge needle (1 in = 25.4 mm), and then left on ice for another 15 min. The homogenate was centrifuged at 430 000 x g for 20 min at 4OC in a Beckman TLA 100.2 rotor. A 100-pL aliquot of Triton-solubilized extract was adjusted to appropriate volume with lysis buffer and then added to 25 pL (packed volume) of gelatin-Sepharose 4B (Pharmacia) or collagen-Sepharose 4B, as required. Binding was accomplished by mixing for 2 h at 4OC. The beads were collected by centrifugation and washed three times with lysis buffer containing 1 M NaCl and finally with 5 mM Hepes (pH 7.5). The bound proteins were eluted by boiling the beads for 3 min ~ I I Laemmli's SDS-polyacrylamide gel electrophoresis sample buffer
Release by low pH or ATP Gelatin-Sepharose beads with bound proteins were washed with 5 mM Hepes buffer and incubated with 50 mM sodium citrate (pH 5.5) on ice for 5 min or with 1 mM ATP and 1 mM MgCl, in 5 mM Hepes (pH 7.5) at room temperature for 10 min. At the end of the treatment, beads were recovered by centrifugation. Proteins left bound to the beads were released by heating with Laemmli sample buffer in a boiling water bath for 3 min and analyzed by SDS-PAGE under reducing conditions. Gels were stained, destained, soaked in EN~HANCE,dried, and exposed to X-ray film at -70°C with an intensifying screen. Immunoprecipitation 3 5 ~ - ~ a b e l l cell e d lysates were preincubated with protein A - Sepharose to reduce nonspecific binding. The clarified lysates were then incubated with a polyclonal antibody to GRP78 (1 :SO dilution) on ice for 90 min. Protein A - Sepharose beads were added and allowed to incubate for a further 30 min. The beads were washed three times with buffer (10 mM Tris-HC1 (pH 7.5) containing 1% Triton X-100,0.6 M NaCI, 0.5 mM phenylmethylsulfonyl fluoride, and 2 pg leupeptin/mL) and finally with 5 mM Hepes (pH 7.4). Bound proteins were released as described before and analyzed by SDS-PAGE under reducing conditions. Immunoblotting was performed as previously described (Cates et al. 1987).
Results Induction of proteins under stress conditions When L6 myoblasts were subjected to environmental insults, induction of the major stress proteins was evident (Fig. 1). Treatment at elevated temperatures (42 or 44°C) resulted in increased synthesis of proteins in the 55-65, 70, and 90-1 10 kDa regions of SDS gels (lanes 1-3). In addition, however, a band at 46 kDa was increased. Exposure to arsenate or arsenite (two inducers with effects similar to heat shock) increased expression of the higher molecular mass proteins, but not the 46-kDa protein (lanes 4 and 5). These results were similar to earlier work on myoblasts (Atkinson
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FIG.4. Peptide mapping of gelatin-bound 78-kDa protein and proteins in gel slices were GRP78. The [35~]methionine-labelled cleaved with chymotrypsin (0.5 mg/mL) as described (Munro and Pelham 1986), run on SDS-PAGE, and autoradiographed. Lane 1, GRP78; lane 2, 78-kDa protein.
FIG. 3. Gelatin-binding proteins in tunicamycin-treated L6 cells. Gelatin-binding proteins in extracts from myoblasts treated without (lane 1) or with (lane 2) tunicamycin and labelled with [35~]methionine were run on SDS-PAGE and autoradiographed.
1981; Kim et al. 1983). When cells were grown in the presence of tunicamycin, which induces GRPs, bands at 78 and 100 kDa became prominent, but no change in the 46-kDa region was apparent. A band seen at 160 kDa in lane 6 was not reproducibly induced in other experiments. Hence the pattern of stress protein expression in the L6 line was very similar to other cell types, with the exception of the prominent 46-kDa protein. To determine if the 46-kDa protein induced by heat was the GP46 protein which we have previously identified (Cates et al. 1984), monoclonal antibodies were used. Extracts of L6 cells heat treated for several lengths of time were immunoblotted (Fig. 2). The results show that GP46 levels were increased at 6 h (lanes 1 and 2) and remained elevated (2.5-fold increase) over 24 h of growth at 42OC. Higher temperatures resulted in a more rapid increase, but to a similar final level. This increase was not due to a change in the protein's turnover rate, which was unchanged in heat-shocked
cells (results not shown). Thus GP46 is an HSP and accounts for at least some of the increase in the 46-kDa region seen in Fig. 1. Binding of GRP78 and GP46 to gelatin It is known that GRP78 binds to some aberrant proteins in the ER (Kozutsumi et al. 1988; Sharma et al. 1985) and probably either helps in the protection or renaturation of the malfolded proteins. Since GP46 is in the ER and is a glycoprotein, we wished to examine whether GRP78 is able to associate with nonglycosylated GP46. To isolate possible GRP78-GP46 complexes, L6 cell extracts were treated with gelatin-Sepharose beads, because we had earlier demonstrated (Cates et al. 1987) that GP46 is a gelatin-binding protein. Four major proteins from untreated L6 myoblasts bound to gelatin (Fig. 3). The band at around 220-250 kDa comigrated with fibronectin, while the band at 46 kDa was GP46. Additional bands at 78 and 72 kDa were also present. In the presence of tunicamycin the 78-kDa band increased and the molecular mass of GP46 was reduced to 42 kDa, because the newly synthesized protein remained unglycosylated. One possible explanation of these results was that the 78-kDa protein was induced, as expected of GRP78. To demonstrate that the 78-kDa protein was indeed GRP78, the protein was excised from a gel and mapped by partial proteolysis. Authentic, labelled GRP78 was isolated from myoblasts by immunoprecipitation with a specific antibody for comparison. The two proteins gave virtually identical patterns of peptides after chymotryptic cleavage (Fig. 4). The question then arose as to whether GRP78 bound by itself to gelatin or as a complex with GP46. To answer this question we exposed the gelatin beads after adsorption of extracts from L6 cells to conditions that selectively eluted the proteins. Proteins that remained bound were separated
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FIG. 5. Selective release of gelatin-bound proteins. GelatinSepharose beads with adsorbed proteins from L6 (lanes 1-3) or tunicamycin-treated L6 (lanes 4-6) were incubated at pH 7.5 (lanes I and 4) or 5.5 (lanes 2 and 5), or with 1 mM ATP and I mM MgCI, (lanes 3 and 6). Proteins that remained bound were eluted with SDS sample buffer, run on SDS-PAGE, and autoradiographed.
by SDS-PAGE (Fig. 5). At pH 5.5, GP46 (or its 42-kDa nonglycosylated form) was eluted from the gelatin without affecting GRP78 (e.g., bound GP46 is decreased in lane 2, after pH 5.5 treatment, compared to lane 1). Conversely, removal of GRP78 did not affect binding of GP46; treatment with ATP decreased the amount of GRP78, but not GP46 remaining (compare lanes 1 and 2). Thus both proteins must independently bind to gelatin. Since GRP78 is thought to recognize malfolded proteins, it may bind to gelatin because it is a denatured form of collagen. In accord with this possibility, we have found that GRP78 does not bind to collagen, whereas GP46 does (results not shown).
Discussion The synthesis of specific proteins in response to stress in L6 myoblasts is similar to other cell types that have been studied. In particular a 78-kDa protein is the well-known GRP78; it is induced slightly by heat and extensively by tunicamycin or glucose deprivation. The relationship of GP46 to other described proteins is less clear. Several of its properties suggest a close parallel with a 47-kDa heatinducible protein from chick cells (Nagata et al. 1986). Both proteins are residents of the ER and bind to collagen and gelatin in a pH-dependent manner. On the other hand GP46
and the 47-kDa protein have different peptide maps and N-terminal sequences (Nagata et al. 1988; our unpublished results). GP46 is identical by peptide mapping to the protein colligin described in several mouse cell lines (Kurkinen et al. 1984). Hence GP46 represents the rat form of colligin and the chick 47-kDa protein may be in the same family. Our results in rat myoblasts suggest that these proteins will be heat inducible in many cell types. There are several similarities between GRP78 and GP46, possibly reflecting a similar overall function although the differences between them imply distinct actions. The binding of both proteins to the unfolded form of collagen gives them the potential to play a role in the collagen assembly process. Their location in the ER where collagen folding takes place, induction by conditions that denature proteins, and the probable role of GRP78 in IgG assembly also lend support to this idea. Further, the assembly of collagen is a complex process involving a variety of modifications and interactions with enzymes before a stable structure is attained. It would not be surprising to find additional proteins involved in folding. Both GP46 and GRP78 are present in nonstressed cells and may function in the course of normal biosynthesis, as well as after stress. The tissue distribution of GP46, which is coincident with collagen production (D. Nandan, G. Cates, E. Ball, and B. Sanwal, in preparation), implicates the protein in a collagen-specific role, in contrast to the more general role of GRP78. Procollagen assembly is likely to be even more complex than previously thought. The actual function of the stress proteins is not clear at this point, but several possibilities are evident. In line with its proposed action during IgG synthesis, GRP78 may be responsible for binding to individual procollagen chains before assembly into a triple helical molecule. This may prevent the formation of insoluble aggregates or keep the chains in an assembly-competent state by some other means. Release of GRP78 by ATP suggests some active function; however, it is currently unknown if the bound protein suffers any modification or if ATP serves simply to periodically release the stress protein and expose the assembly site for interaction with other subunits (Kassenbrock and Kelley 1989). Further work on these possibilities will require isolation of large amounts of the protein. Release of GP46 by low pH points to a different role for this protein: delivery of gelatin or collagen to an acidic compartment. The pH in the cis golgi and in lysosomes is comparatively low, and GP46 could plausibly be involved in transferring gelatin and (or) procollagen to either place. It is known that a large fraction of procollagen chains synthesized are degraded without being secreted (Berg et al. 1980). Thus GP46 may be associated with selection of assembled procollagen for secretion or malfolded procollagen for degradation. As GP46 is better characterized, its properties and binding affinities may point in one of these directions. The question of what determinants on gelatin are recognized by GP46 and GRP78 is not yet answered. GP46 (but not GRP78) can be eluted using the short peptide ArgGly-Asp-Ser (Nandan et al. 1988a), suggesting that a particular sequence of amino acids is a recognition site. GRPX, on the other hand, binds to a spectrum of proteins, possibly recognizing glycosylation sites (Chang et al. 1987) or exposed hydrophobic areas (Munro and Pelham 1986). More recent work has implicated a mobile, extended polypeptide
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NANDAN ET AL.
chain as important (Flynn et al. 1989) and binding t o gelatin is more in accord with this possibility. Hence t h e two proteins seem t o recognize quite different features of gelatin, again pointing t o different roles for them in procollagen assembly o r degradation. A major use of t h e findings of this research is likely t o be in the purification of GRP78. Release by A T P f r o m t h e easily available gelatin-Sepharose should make isolation o f the protein relatively straightforward a n d preliminary studies bear this out (our unpublished results). W e anticipate that this will allow resolution of some o f the questions posed by this intriguing protein.
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R.B. 1988. Heavy chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature (London), 333: 90-93. KIM, Y.-J., SHUMAN,J., SETTE,M., and PRZYBYLA, A. 1983. Arsenate induces stress proteins in cultured rat myoblasts. J. Cell Biol. 96: 393-400. KOCH,G.L.E. 1987. Reticuloplasmins: a novel group of proteins in the endoplasmic reticulum. J. Cell Sci. 87: 491-492. KOZUTSUMI,Y., SEGAL,M., NORMINGTON, K., GETHING,M.-J., and SAMBROOK, J. 1988. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucoseregulated proteins. Nature (London), 332: 462-464. KURKINEN, M., TAYLOR, A., GARRELS, J.I., and HOGAN,B.L.M. 1984. Cell surface associated proteins which bind native type IV collagen or gelatin. J. Biol. Chem. 259: 5915-5922. Acknowledgements LAEMMLI,U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), W e thank Dr. H. Pelham for his gift o f anti-GRP78. This 227: 680-685. research was supported by the Medical Research Council LEE, A.S. 1987. Coordinated regulation of a set of genes by of Canada. -glucose and calcium ionouhores in mammalian cells. Trends Biochem. Sci. 12: 20-23. ATKINSON, B.G. 1981. Synthesis of heat shock proteins by cells LEE, A.S., BELL, J., and TING, J., 1984. Biochemical undergoing myogenesis. J. Cell Biol. 89: 666-673. characterization of the 94- and 78-kilodalton glucose regulated BERG,R.A., SCHWARTZ, M.L., and CRYSTAL, R.G. 1980. Regulaproteins in hamster fibroblasts. J. Biol. Chem. 259: 4616-4621. tion of the production of secretory proteins: Intracellular MUNRO,S., and PELHAM,H.R.B. 1986. An hsp 70-like protein degradation of newly synthesized "defective" collagen. Proc. in the ER: identity with the 78 Kd glucose regulated protein and Natl. Acad. Sci. U.S.A. 77: 4746-4750. immunoglobulin heavy chain binding protein. Cell, 46: 291-300. BOLE,D.G., HENDERSHOT, L.M., and KEARNEY, J.F. 1986. Post -1987. A C-terminal signal prevents secretion of luminal ER translational association of immunoglobulin heavy chains in proteins. Cell, 48: 899-907. nonsecreting and secreting hybridomas. J. Cell Biol. 102: NAGATA,K., SAGA,S., and YAMADA,K.M. 1986. A major col1558-1566. lagen binding protein of chick embryo fibroblasts is a novel heat CATES, G.A., BRICKENDEN, A.M., and SANWAL,B.D. 1984. shock protein. J. Cell Biol. 103: 223-229. Possible involvement of a cell surface glycoprotein in the dif-1988. Characterization of a novel transformation-sensitive ferentiation of skeletal myoblasts. J. Biol. Chem. 259: 2646-2650. heat shock protein (HSP 47) that binds to collagen. Biochem. CATES,G.A., NANDAN,D., BRICKENDEN, A.M., and SANWAL, Biophys. Res. Commun. 153: 428-434. B.D. 1987. Differentiation defective mutants of skeletal NANDAN,D., CATES,G.A., BALL, E.H., and SANWAL,B.D. myoblasts altered in a gelatin-binding glycoprotein. Biochem. 1988a. A collagen binding protein involved in the differentiation Cell Biol. 65: 767-775. of myoblasts recognizes the arg-gly-asp sequence. Exp. Cell Res. CHANG,S.C., WOODEN,S.K., NAKAKI,T., KIM, Y.K., LIN, 179: 289-297. A.Y ., KUNG,L., ATTENELLO,J.W., and LEE, A.S. 1987. Rat NANDAN,D., ZEUNER, E.P., BRICKENDEN, A.. BALL,E.H., and gene encoding the 78-kDa glucose regulated protein GRP78: Its SANWAL,B.D. 1988b. Characterization and cloning of GP46, regulatory sequences and the effect of protein glycosylation on a collagen binding glycoprotein involved in myoblast differenits expression. Proc. Natl. Acad. Sci. U.S.A. 84: 680-684. tiation. Proceedings of the IV International Congress of Cell DESHAIES, R.J., KOCH,B.D.. and SCHEKMAN. R. 1988. The role Biology, Montreal, Que. p. 184. of stress proteins in membrane biogenesis. rends Biochem. Sci. PELHAM,H.R.B. 1986. Speculation on the functions of the major 13: 384-388. heat shock and glycose-regulated proteins. Cell, 46: 959-961. FLYNN,G.C., CHAPPELL,T.G., and ROTHMAN,J.E. 1989. SHARMA,S., RODGERS, L., BRANDSMA, J., GETHING,M.-J., and Peptide binding and release by proteins implicated as catalysts SAMBROOK,J. 1985. SV40 T antigen and the exocytotic of protein assembly. Science (Washington, D.C.). 245: 385-390. pathway. EMBO J. 4: 1479-1489. KASSENBROCK, C.K., and KELLY,R.B. 1989. Interaction of heavy YAFFE,D. 1%8. Retention of differentiation potentialities during chain binding protein (BiP/GRP78) with adenine nucleotides. prolonged cultivation of myogenic cells. Proc. Natl. Acad. Sci. EMBO J. 8: 1461-1467. U.S.A. 61: 477-483. KASSENBROCK, C.K., GARCIA,P.D., WALTER,P., and KELLY,
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Department of Biochemistry, University of Western Ontario, London, Ont., Canada N6A 5CI Received December 15, 1989 NANDAN,D., BALL,E. H., and SANWAL, B. D. 1990. Two stress proteins of the endoplasmic reticulum bind denatured collagen. Biochem. Cell Biol. 68: 1057-1061. A differentiation-related gelatin-binding 46 kilodalton (kDa) glycoprotein in myoblasts (GP46, colligin) shares several properties with the 78-kDa glucose-regulated protein (GRP78), including location in the endoplasmic reticulum and related C-terminal sequences. These similarities extend to stress inducibility, since we find that GP46 is a heat-shock protein; its synthesis is elevated at 42OC, resulting in a two- to three-fold increase in protein level. Further, GRP78 is a gelatin-binding protein; together with GP46 it is retained on gelatin-Sepharose beads. GRP78 and GP46 do not interact; each protein can be individually eluted, GP46 at low pH and GRP78 by ATP. These results suggest that the proteins have distinct roles in the synthesis of collagen and point to a simple method for purification. Key words: stress proteins, collagen-binding proteins, endoplasmic reticulum, 78-kilodalton glucose-regulated protein, 46-kilodalton glycoprotein.
NANDAN. D., BALL,E. H., et SANWAL,B. D. 1990. Two stress proteins of the endoplasmic reticulum bind denatured collagen. Biochem. Cell Biol. 68 : 1057-1061. Dans les myoblastes, une glycoproteine (GP46, colligine) de 46 kilodaltons (kDa), liant la gklatine et relike a la differenciation, partage plusieurs proprihtks avec la protkine de 78 kDa contr81Ce par le glucose (GRP78), dont la localisation dans le reticulum endoplasmique et les sequences C-terminales apparentees. Ces similitudes comprennent Cgalement l'inductibilite au stress, car nous trouvons que la GP46 est une proteine de choc thermique; a 42°C. sa synthtse elevte augmente le taux protkique de deux a trois fois. De plus, la GRP78 est une protkine liant la gelatine; les deux prottines, GRP78 et GP46, sont retenues sur les billes de gelatine-Stpharose. La GRP78 ne rCagit pas avec la GP46; chacune peut Etre Clute individuellement, la GP46 a faible pH et la GRP78 par I'ATP. Ces rksultats sugghent que ces proteines ont des r6les distincts dans la synthhse du collagtne et laissent prksager une methode simple de purification. Mots cl&s : pprteines de stress, protkines laissent le collagtne, reticulum endoplasmique, proteine de 78 kilodaltons contr81Ce par le glucose, glycoproteine de 46 kilodaltons. [Traduit par la revue]
Eucaryotic cells respond to stress conditions, such as heat shock, glucose deprivation, and certain chemical treatments by overproducing a set of stress proteins (for reviews, see Deshaies et al. 1988; Lee 1987; Pelham 1986). Two major classes of these proteins have been identified: the GRPs are localized to the ER (Pelham 1986; Munro and Pelham 1987) and the HSPs are mostly cytoplasmic (Pelham 1986). This division is not absolute; some GRPs are slightly heat inducible (Lee et al. 1984). Also, an apparent exception with regard to the localization of a HSP is a 47-kDa protein from chicken embryo fibroblasts, which despite its location in the ER has been found to be inducible by heat shock (Nagata et al. 1986). The role of stress proteins is not known for certain, but the consensus is emerging (Pelham 1986) that they participate in the refolding of denatured proteins or the initial folding of newly synthesized proteins. Best understood are GRP78 (also known as BiP) and the related HSP70. In lymphoid cells GRP78 associates with newly synthesized immunoglobulin heavy chains until their assembly with light chains and probably thus prevents the transport of unassembled heavy chains from the ER. In nonlymphoid cells, which do not produce immunoglobulins, GRP78, an inducible protein, associates with abnormal proteins (Lee 1987; Bole et al. 1986; Kozutsumi et al. 1988; Kassenbrock et al. 1988) and probABBREVIATIONS: GP46, 46-kilodalton glycoprotein (colligin); GRP78, 78-kDa glucose-regulated protein; ER, endoplasmic reticulum; HSPs, heat-shock-inducible proteins; BiP, binding protein; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; kDa, kilodalton(s); IgG, immunoglobulin G. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprim6 au Canada
ably helps in their renaturation. Both classes of stress protein probably function to limit damage owing to protein denaturation. While most attention has been devoted to GRP78, next to nothing is known regarding the action of other stress proteins that are present in the lumen of the ER, viz., GRP94 (Lee et al. 1984; Koch 1987) and GP47 (Nagata et al. 1986). During a study of the role of a GP46 in the differentiation of rat skeletal myoblasts (Cates et al. 1984), we discovered that it was localized in the lumen of the ER (Nandan et al. 1988a) and had a RDEL sequence at the C-terminus, as determined by sequencing a partial cDNA clone (Nandan et al. 1988b). A homologous sequence, KDEL, is shared by GRP78 and GRP94 (Munro and Pelham 1987) and may be responsible for retention in the ER. These similarities in structure and location suggested related functions and prompted us to compare them in more detail. In the following report, we demonstrate that in skeletal myoblasts GP46 is a stress protein and GRP78 shares with it the property of binding to gelatin. The functions of the two stress proteins, however, seem to be quite different. Materials and methods Materials A highly myogenic subclone of the rat myoblast cell line, L6, first isolated by Yaffe (1968) was used. A monoclonal antibody against GP46 was obtained as described earlier (Cates et al. 1987). A polyclonal antibody against GRP78 was kindly provided by Dr. H. Pelham (U.K.). Cell culture and metabolic labelling Cells were routinely cultured in minimum essential medium supplemented with 50 pg gentamycin/mL, 16 mM glucose, and 10% horse serum. For tunicamycin treatment and metabolic labelling,
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FIG. 2. Induction of GP46 at 42OC. After 48 h of growth, cultures were transferred to 42OC for various lengths of time. Cells were then lysed in SDS sample buffer, run on SDS gels, and immunoblotted using monoclonal anti-GP46.Odd-numbered lanes are from controls at 37OC; even numbered lanes are from those at 42°C. Lanes 1 and 2, after 6 h of heat treatment; lanes 3 and 4, after 8 h; lanes 5 and 6, after 13 h; lanes 7 and 8, after 24 h. Note that the level of GP46 in control cells increases as the cells approach fusion. (Laemmli 1970). Collagen-Sepharose was made by coupling rat type I collagen to cyanogen bromide activated Sepharose (Pharmacia) according to the manufacturer's instructions.
FIG. 1. Protein synthesis in stressed L6 myoblasts. Cells were treated for 4 h under various conditions: at 37 (lane l), 42 (lane 2), or 44°C (lane 3), or with 0.1 mM sodium arsenate (lane 4), sodium arsenite (lane 5), or 5 p tunicamycin/mL (lane 6). After a further 1 h of labelling with [ 3$ Slmethionine, cultures were lysed in SDS sample buffer and equal amounts of TCA-precipitable radioactivity were loaded on an 8-13% gradient gel. An autoradiograph of the gel is shown. cells were plated at an initial density of 1.5 x 10' cells/60-mm plate and grown for 72 h. After this period, growth medium was replaced with fresh medium containing 5 pg tunicarnycin/mL. After 5 h of preincubation with tunicamycin, cells were labelled for 2 h with 0.1 mCi [35~]methionine/m~ (1 Ci = 36 GBq) in methioninefree medium containing the desired amount of tunicamycin.
Isolation of gelatin- or collagen-binding proteins Cells were rinsed three times with cold PBS and extracted with 2 mL lysis buffer (10 mM Tris-HC1 (pH 7 . 9 , 0.15 M NaCl, 1% Triton X-100, 2 pg leupeptin/mL, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM EGTA). After 5 min on ice, the cells were scraped, passed three times through a 0.5-in 27-gauge needle (1 in = 25.4 mm), and then left on ice for another 15 min. The homogenate was centrifuged at 430 000 x g for 20 min at 4OC in a Beckman TLA 100.2 rotor. A 100-pL aliquot of Triton-solubilized extract was adjusted to appropriate volume with lysis buffer and then added to 25 pL (packed volume) of gelatin-Sepharose 4B (Pharmacia) or collagen-Sepharose 4B, as required. Binding was accomplished by mixing for 2 h at 4OC. The beads were collected by centrifugation and washed three times with lysis buffer containing 1 M NaCl and finally with 5 mM Hepes (pH 7.5). The bound proteins were eluted by boiling the beads for 3 min ~ I I Laemmli's SDS-polyacrylamide gel electrophoresis sample buffer
Release by low pH or ATP Gelatin-Sepharose beads with bound proteins were washed with 5 mM Hepes buffer and incubated with 50 mM sodium citrate (pH 5.5) on ice for 5 min or with 1 mM ATP and 1 mM MgCl, in 5 mM Hepes (pH 7.5) at room temperature for 10 min. At the end of the treatment, beads were recovered by centrifugation. Proteins left bound to the beads were released by heating with Laemmli sample buffer in a boiling water bath for 3 min and analyzed by SDS-PAGE under reducing conditions. Gels were stained, destained, soaked in EN~HANCE,dried, and exposed to X-ray film at -70°C with an intensifying screen. Immunoprecipitation 3 5 ~ - ~ a b e l l cell e d lysates were preincubated with protein A - Sepharose to reduce nonspecific binding. The clarified lysates were then incubated with a polyclonal antibody to GRP78 (1 :SO dilution) on ice for 90 min. Protein A - Sepharose beads were added and allowed to incubate for a further 30 min. The beads were washed three times with buffer (10 mM Tris-HC1 (pH 7.5) containing 1% Triton X-100,0.6 M NaCI, 0.5 mM phenylmethylsulfonyl fluoride, and 2 pg leupeptin/mL) and finally with 5 mM Hepes (pH 7.4). Bound proteins were released as described before and analyzed by SDS-PAGE under reducing conditions. Immunoblotting was performed as previously described (Cates et al. 1987).
Results Induction of proteins under stress conditions When L6 myoblasts were subjected to environmental insults, induction of the major stress proteins was evident (Fig. 1). Treatment at elevated temperatures (42 or 44°C) resulted in increased synthesis of proteins in the 55-65, 70, and 90-1 10 kDa regions of SDS gels (lanes 1-3). In addition, however, a band at 46 kDa was increased. Exposure to arsenate or arsenite (two inducers with effects similar to heat shock) increased expression of the higher molecular mass proteins, but not the 46-kDa protein (lanes 4 and 5). These results were similar to earlier work on myoblasts (Atkinson
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FIG.4. Peptide mapping of gelatin-bound 78-kDa protein and proteins in gel slices were GRP78. The [35~]methionine-labelled cleaved with chymotrypsin (0.5 mg/mL) as described (Munro and Pelham 1986), run on SDS-PAGE, and autoradiographed. Lane 1, GRP78; lane 2, 78-kDa protein.
FIG. 3. Gelatin-binding proteins in tunicamycin-treated L6 cells. Gelatin-binding proteins in extracts from myoblasts treated without (lane 1) or with (lane 2) tunicamycin and labelled with [35~]methionine were run on SDS-PAGE and autoradiographed.
1981; Kim et al. 1983). When cells were grown in the presence of tunicamycin, which induces GRPs, bands at 78 and 100 kDa became prominent, but no change in the 46-kDa region was apparent. A band seen at 160 kDa in lane 6 was not reproducibly induced in other experiments. Hence the pattern of stress protein expression in the L6 line was very similar to other cell types, with the exception of the prominent 46-kDa protein. To determine if the 46-kDa protein induced by heat was the GP46 protein which we have previously identified (Cates et al. 1984), monoclonal antibodies were used. Extracts of L6 cells heat treated for several lengths of time were immunoblotted (Fig. 2). The results show that GP46 levels were increased at 6 h (lanes 1 and 2) and remained elevated (2.5-fold increase) over 24 h of growth at 42OC. Higher temperatures resulted in a more rapid increase, but to a similar final level. This increase was not due to a change in the protein's turnover rate, which was unchanged in heat-shocked
cells (results not shown). Thus GP46 is an HSP and accounts for at least some of the increase in the 46-kDa region seen in Fig. 1. Binding of GRP78 and GP46 to gelatin It is known that GRP78 binds to some aberrant proteins in the ER (Kozutsumi et al. 1988; Sharma et al. 1985) and probably either helps in the protection or renaturation of the malfolded proteins. Since GP46 is in the ER and is a glycoprotein, we wished to examine whether GRP78 is able to associate with nonglycosylated GP46. To isolate possible GRP78-GP46 complexes, L6 cell extracts were treated with gelatin-Sepharose beads, because we had earlier demonstrated (Cates et al. 1987) that GP46 is a gelatin-binding protein. Four major proteins from untreated L6 myoblasts bound to gelatin (Fig. 3). The band at around 220-250 kDa comigrated with fibronectin, while the band at 46 kDa was GP46. Additional bands at 78 and 72 kDa were also present. In the presence of tunicamycin the 78-kDa band increased and the molecular mass of GP46 was reduced to 42 kDa, because the newly synthesized protein remained unglycosylated. One possible explanation of these results was that the 78-kDa protein was induced, as expected of GRP78. To demonstrate that the 78-kDa protein was indeed GRP78, the protein was excised from a gel and mapped by partial proteolysis. Authentic, labelled GRP78 was isolated from myoblasts by immunoprecipitation with a specific antibody for comparison. The two proteins gave virtually identical patterns of peptides after chymotryptic cleavage (Fig. 4). The question then arose as to whether GRP78 bound by itself to gelatin or as a complex with GP46. To answer this question we exposed the gelatin beads after adsorption of extracts from L6 cells to conditions that selectively eluted the proteins. Proteins that remained bound were separated
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FIG. 5. Selective release of gelatin-bound proteins. GelatinSepharose beads with adsorbed proteins from L6 (lanes 1-3) or tunicamycin-treated L6 (lanes 4-6) were incubated at pH 7.5 (lanes I and 4) or 5.5 (lanes 2 and 5), or with 1 mM ATP and I mM MgCI, (lanes 3 and 6). Proteins that remained bound were eluted with SDS sample buffer, run on SDS-PAGE, and autoradiographed.
by SDS-PAGE (Fig. 5). At pH 5.5, GP46 (or its 42-kDa nonglycosylated form) was eluted from the gelatin without affecting GRP78 (e.g., bound GP46 is decreased in lane 2, after pH 5.5 treatment, compared to lane 1). Conversely, removal of GRP78 did not affect binding of GP46; treatment with ATP decreased the amount of GRP78, but not GP46 remaining (compare lanes 1 and 2). Thus both proteins must independently bind to gelatin. Since GRP78 is thought to recognize malfolded proteins, it may bind to gelatin because it is a denatured form of collagen. In accord with this possibility, we have found that GRP78 does not bind to collagen, whereas GP46 does (results not shown).
Discussion The synthesis of specific proteins in response to stress in L6 myoblasts is similar to other cell types that have been studied. In particular a 78-kDa protein is the well-known GRP78; it is induced slightly by heat and extensively by tunicamycin or glucose deprivation. The relationship of GP46 to other described proteins is less clear. Several of its properties suggest a close parallel with a 47-kDa heatinducible protein from chick cells (Nagata et al. 1986). Both proteins are residents of the ER and bind to collagen and gelatin in a pH-dependent manner. On the other hand GP46
and the 47-kDa protein have different peptide maps and N-terminal sequences (Nagata et al. 1988; our unpublished results). GP46 is identical by peptide mapping to the protein colligin described in several mouse cell lines (Kurkinen et al. 1984). Hence GP46 represents the rat form of colligin and the chick 47-kDa protein may be in the same family. Our results in rat myoblasts suggest that these proteins will be heat inducible in many cell types. There are several similarities between GRP78 and GP46, possibly reflecting a similar overall function although the differences between them imply distinct actions. The binding of both proteins to the unfolded form of collagen gives them the potential to play a role in the collagen assembly process. Their location in the ER where collagen folding takes place, induction by conditions that denature proteins, and the probable role of GRP78 in IgG assembly also lend support to this idea. Further, the assembly of collagen is a complex process involving a variety of modifications and interactions with enzymes before a stable structure is attained. It would not be surprising to find additional proteins involved in folding. Both GP46 and GRP78 are present in nonstressed cells and may function in the course of normal biosynthesis, as well as after stress. The tissue distribution of GP46, which is coincident with collagen production (D. Nandan, G. Cates, E. Ball, and B. Sanwal, in preparation), implicates the protein in a collagen-specific role, in contrast to the more general role of GRP78. Procollagen assembly is likely to be even more complex than previously thought. The actual function of the stress proteins is not clear at this point, but several possibilities are evident. In line with its proposed action during IgG synthesis, GRP78 may be responsible for binding to individual procollagen chains before assembly into a triple helical molecule. This may prevent the formation of insoluble aggregates or keep the chains in an assembly-competent state by some other means. Release of GRP78 by ATP suggests some active function; however, it is currently unknown if the bound protein suffers any modification or if ATP serves simply to periodically release the stress protein and expose the assembly site for interaction with other subunits (Kassenbrock and Kelley 1989). Further work on these possibilities will require isolation of large amounts of the protein. Release of GP46 by low pH points to a different role for this protein: delivery of gelatin or collagen to an acidic compartment. The pH in the cis golgi and in lysosomes is comparatively low, and GP46 could plausibly be involved in transferring gelatin and (or) procollagen to either place. It is known that a large fraction of procollagen chains synthesized are degraded without being secreted (Berg et al. 1980). Thus GP46 may be associated with selection of assembled procollagen for secretion or malfolded procollagen for degradation. As GP46 is better characterized, its properties and binding affinities may point in one of these directions. The question of what determinants on gelatin are recognized by GP46 and GRP78 is not yet answered. GP46 (but not GRP78) can be eluted using the short peptide ArgGly-Asp-Ser (Nandan et al. 1988a), suggesting that a particular sequence of amino acids is a recognition site. GRPX, on the other hand, binds to a spectrum of proteins, possibly recognizing glycosylation sites (Chang et al. 1987) or exposed hydrophobic areas (Munro and Pelham 1986). More recent work has implicated a mobile, extended polypeptide
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NANDAN ET AL.
chain as important (Flynn et al. 1989) and binding t o gelatin is more in accord with this possibility. Hence t h e two proteins seem t o recognize quite different features of gelatin, again pointing t o different roles for them in procollagen assembly o r degradation. A major use of t h e findings of this research is likely t o be in the purification of GRP78. Release by A T P f r o m t h e easily available gelatin-Sepharose should make isolation o f the protein relatively straightforward a n d preliminary studies bear this out (our unpublished results). W e anticipate that this will allow resolution of some o f the questions posed by this intriguing protein.
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R.B. 1988. Heavy chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature (London), 333: 90-93. KIM, Y.-J., SHUMAN,J., SETTE,M., and PRZYBYLA, A. 1983. Arsenate induces stress proteins in cultured rat myoblasts. J. Cell Biol. 96: 393-400. KOCH,G.L.E. 1987. Reticuloplasmins: a novel group of proteins in the endoplasmic reticulum. J. Cell Sci. 87: 491-492. KOZUTSUMI,Y., SEGAL,M., NORMINGTON, K., GETHING,M.-J., and SAMBROOK, J. 1988. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucoseregulated proteins. Nature (London), 332: 462-464. KURKINEN, M., TAYLOR, A., GARRELS, J.I., and HOGAN,B.L.M. 1984. Cell surface associated proteins which bind native type IV collagen or gelatin. J. Biol. Chem. 259: 5915-5922. Acknowledgements LAEMMLI,U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), W e thank Dr. H. Pelham for his gift o f anti-GRP78. This 227: 680-685. research was supported by the Medical Research Council LEE, A.S. 1987. Coordinated regulation of a set of genes by of Canada. -glucose and calcium ionouhores in mammalian cells. Trends Biochem. Sci. 12: 20-23. ATKINSON, B.G. 1981. Synthesis of heat shock proteins by cells LEE, A.S., BELL, J., and TING, J., 1984. Biochemical undergoing myogenesis. J. Cell Biol. 89: 666-673. characterization of the 94- and 78-kilodalton glucose regulated BERG,R.A., SCHWARTZ, M.L., and CRYSTAL, R.G. 1980. Regulaproteins in hamster fibroblasts. J. Biol. Chem. 259: 4616-4621. tion of the production of secretory proteins: Intracellular MUNRO,S., and PELHAM,H.R.B. 1986. An hsp 70-like protein degradation of newly synthesized "defective" collagen. Proc. in the ER: identity with the 78 Kd glucose regulated protein and Natl. Acad. Sci. U.S.A. 77: 4746-4750. immunoglobulin heavy chain binding protein. Cell, 46: 291-300. BOLE,D.G., HENDERSHOT, L.M., and KEARNEY, J.F. 1986. Post -1987. A C-terminal signal prevents secretion of luminal ER translational association of immunoglobulin heavy chains in proteins. Cell, 48: 899-907. nonsecreting and secreting hybridomas. J. Cell Biol. 102: NAGATA,K., SAGA,S., and YAMADA,K.M. 1986. A major col1558-1566. lagen binding protein of chick embryo fibroblasts is a novel heat CATES, G.A., BRICKENDEN, A.M., and SANWAL,B.D. 1984. shock protein. J. Cell Biol. 103: 223-229. Possible involvement of a cell surface glycoprotein in the dif-1988. Characterization of a novel transformation-sensitive ferentiation of skeletal myoblasts. J. Biol. Chem. 259: 2646-2650. heat shock protein (HSP 47) that binds to collagen. Biochem. CATES,G.A., NANDAN,D., BRICKENDEN, A.M., and SANWAL, Biophys. Res. Commun. 153: 428-434. B.D. 1987. Differentiation defective mutants of skeletal NANDAN,D., CATES,G.A., BALL, E.H., and SANWAL,B.D. myoblasts altered in a gelatin-binding glycoprotein. Biochem. 1988a. A collagen binding protein involved in the differentiation Cell Biol. 65: 767-775. of myoblasts recognizes the arg-gly-asp sequence. Exp. Cell Res. CHANG,S.C., WOODEN,S.K., NAKAKI,T., KIM, Y.K., LIN, 179: 289-297. A.Y ., KUNG,L., ATTENELLO,J.W., and LEE, A.S. 1987. Rat NANDAN,D., ZEUNER, E.P., BRICKENDEN, A.. BALL,E.H., and gene encoding the 78-kDa glucose regulated protein GRP78: Its SANWAL,B.D. 1988b. Characterization and cloning of GP46, regulatory sequences and the effect of protein glycosylation on a collagen binding glycoprotein involved in myoblast differenits expression. Proc. Natl. Acad. Sci. U.S.A. 84: 680-684. tiation. Proceedings of the IV International Congress of Cell DESHAIES, R.J., KOCH,B.D.. and SCHEKMAN. R. 1988. The role Biology, Montreal, Que. p. 184. of stress proteins in membrane biogenesis. rends Biochem. Sci. PELHAM,H.R.B. 1986. Speculation on the functions of the major 13: 384-388. heat shock and glycose-regulated proteins. Cell, 46: 959-961. FLYNN,G.C., CHAPPELL,T.G., and ROTHMAN,J.E. 1989. SHARMA,S., RODGERS, L., BRANDSMA, J., GETHING,M.-J., and Peptide binding and release by proteins implicated as catalysts SAMBROOK,J. 1985. SV40 T antigen and the exocytotic of protein assembly. Science (Washington, D.C.). 245: 385-390. pathway. EMBO J. 4: 1479-1489. KASSENBROCK, C.K., and KELLY,R.B. 1989. Interaction of heavy YAFFE,D. 1%8. Retention of differentiation potentialities during chain binding protein (BiP/GRP78) with adenine nucleotides. prolonged cultivation of myogenic cells. Proc. Natl. Acad. Sci. EMBO J. 8: 1461-1467. U.S.A. 61: 477-483. KASSENBROCK, C.K., GARCIA,P.D., WALTER,P., and KELLY,
